Much attention has been paid recently to RNA interference (RNAi), a technique in which exogenous, double-stranded RNAs (dsRNAs) are introduced into a cell to specifically destroy a particular mRNA or block its expression, thereby diminishing or abolishing gene expression (A. Fire et al., “Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans,” Nature, 391:806-11, 1998). Specific types of RNAs, such as small interfering RNAs (siRNAs) and micro interfering RNAs (miRNAs) have been shown to inhibit expression of a number of specific genes effectively and the technique has proven effective in Drosophila, Caenorhabditis elegans, plants, and recently, in mammalian cell cultures (S. M. Elbashir et al., “Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells,” Nature, 411:494-8, 2001). Because small interfering RNA molecules are directed to a specific target and thereby silence a specific gene, they have been suggested to be useful in treatment of diseases as well as for screening new pharmaceuticals and disease mechanisms for pharmaceutical target determination. However, while a number of applications, both therapeutic and screening methods, have been suggested, delivery of RNA interfering agents, including siRNAs and miRNAs, into cells has proven to be the bottleneck.
Currently known methods to deliver RNA interference into cells include chemical transfection using lipid-based, amine-based and polymer-based techniques, and combinations thereof (see, for example, products from Ambion Inc., Austin, Tex.; and Novagen, EMD Biosciences, Inc, an Affiliate of Merck KGaA, Darmstadt, Germany). Unfortunately, efficient transfer of RNA interfering agents, including siRNAs into primary cells by chemical transfection seems to be restricted to a few cell types (Ovcharenko D (2003) “Efficient delivery of siRNAs to human primary cells.” Ambion TechNotes 10 (5): 15-16).
Other described ways to deliver siRNAs include expressing short hairpin RNA molecules from vectors, such as lentiviral constructs, and introducing siRNA molecules into cells using electroporation. However, feline FIV lentivirus vectors which are based on the feline immunodeficiency virus (FIV) retrovirus and the HIV lentivirus vector system, which is base on the human immunodeficiency virus (HIV), carry with them problems related to permanent integration. Electroporation is often a relatively harsh treatment and cannot generally be used to deliver siRNAs into cells in vivo.
An additional problem with all the traditional gene delivery methods discussed above for the use of delivering RNA interference is that they target all cells non-specifically. Therefore, it would be useful to develop gene delivery methods that could be targeted to specific cells thereby minimizing or avoiding potential side effects caused by delivery of RNA interference into non-target cells. Additionally, effective interference RNA delivery methods that could avoid viral vectors and could be used for both in vivo and in vitro delivery of RNA interference, including siRNA, would be desirable.
Moreover, several cell types have proven extremely difficult to transduce with siRNAs using traditional vectors, including viral vectors, liposomes and the like. Such cell types include immune system cells such as lymphocytes and dendritic cells, and stem cells.
One such cell type is T cells. Accordingly, the evaluation of the therapeutic potential of siRNA for HIV infection has been hampered by the challenges of delivery to T cells and till recently, the lack of an effective small animal model. The toxicity and frequent treatment failures associated with HAART has focused attention on the potent gene silencing mechanism of RNAi as an alternate treatment strategy for HIV infection1-4. Several in vitro studies have shown the potential of RNAi for effective suppression of HIV infection in cell lines and primary human T cells and macrophages, the prime targets of HIV5-9. However for actual therapeutic use, many parameters such as effective siRNA delivery to susceptible cells, antiviral efficacy and toxicity need to be tested in vivo. The recently described IL2 receptor common gamma chain null (IL2rγ−/−) strains of immunodeficient mice allow efficient reconstitution of human immune cells making them amenable for HIV infection10-13, but siRNA delivery to primary human T cells still remains a hurdle. Thus, so far no study has actually tested the potential of siRNA treatment to suppress HIV infection in vivo.
Therefore, to utilize fully the potential in treatment and drug screening of the discovered RNA interference, including siRNAs, it is necessary to develop ways to deliver siRNAs into cells both in vitro and in vivo.